Chikungunya fever swept across many South and South-east Asian countries, following extensive outbreaks in the Indian Ocean Islands in 2005. However, molecular epidemiological data to explain the recent spread and evolution of Chikungunya virus (CHIKV) in the Asian region are still limited. This study describes the genetic Characteristics and evolutionary relationships of CHIKV strains that emerged in Sri Lanka and Singapore during 2006-2008. The viruses isolated in Singapore also included those imported from the Maldives (n=1), India (n=2) and Malaysia (n=31). All analysed strains belonged to the East, Central and South African (ECSA) lineage and were evolutionarily more related to Indian than to Indian Ocean Islands strains. Unique genetic characteristics revealed five genetically distinct subpopulations of CHIKV in Sri Lanka and Singapore, which were likely to have emerged through multiple, independent introductions. The evolutionary network based on E1 gene sequences indicated the acquisition of an alanine to valine 226 substitution (E1-A226V) by virus strains of the Indian sublineage as a key evolutionary event that contributed to the transmission and spatial distribution of CHIKV in the region. The E1-A226V substitution was found in 95.7 % (133/139) of analysed isolates in 2008, highlighting the widespread establishment of mutated CHIKV strains in Sri Lanka, Singapore and Malaysia. As the E1-A226V substitution is known to enhance the transmissibility of CHIKV by Aedes albopictus mosquitoes, this observation has important implications for the design of vector control strategies to fight the virus in regions at risk of chikungunya fever.